DESCRIPTION (Taken directly from the application) Long term treatment of cystic fibrosis (CF) through gene therapy will require that the transgene be stably maintained within the transduced cell population. Further, the vector should not express viral antigens which will elicit an immune response leading to the destruction of the transduced cells. One of the most promising viral vector systems currently being developed for the treatment of CF is based on recombinant adeno-associated virus (rAAV). The AAV genome contains terminal repeat (TR) sequences at each end which promote the integration of the genome into the host chromosome. Recombinant AAV vectors offer two advantages over other viral vector systems: they can be maintained in the integrated state for the lifetime of the transduced cell and its progeny; and, the vectors can be constructed such that no viral genes remain to be expressed and to elicit an immune response. However, rAAV vectors which retain the viral rep gene are specifically integrated into a site on human chromosome 19. This represents an important safety feature in vectors with this property. Disadvantages of rAAV vectors are a stringent size limitation for packaging and difficulty in growing high titer virus stocks of suitable purity. We have created a new DNA vector, double D, which utilizes modified AAV TRs to promote integration but can be propagated as a plasmid in bacteria. This hybrid vector thus solves the problems of transgene coding capacity and production of large quantities of purified vector. We propose to develop methods for the efficient delivery of this vector into cultured cells, including airway epithelial cells, using liposome mediated transfection. This work will also entail the transient co-delivery of the AAV Rep protein in bans such that integration of the vector will be directed to the chromosome 19 site. Finally, we will utilize the double D vector to deliver the CFTR gene into cells and determine the magnitude and duration of its Expression.